[The functional differences of macrophages derived from murine bone marrow and peritoneal cavity].

Objective To compare the functional differences between bone marrow derived macrophages and peritoneal macrophages, which may provide the basis for the selection of macrophages in immunological research and immunoregulatory drug evaluation. Methods Marophage colony-stimulating factor (M-CSF) was used to induce the differentiation of bone marrow monocytes into macrophages, and thioglycolate medium was used to induce peritonitis to obtain peritoneal macrophages. After both macrophages being stimulated by zymosan, LPS, R848 and CpG respectively, mRNA levels of tumor necrosis factor α(TNF-α), interleukin 6(IL-6), macrophage inflammatory protein 1α(MIP-1α), monocyte chemoattractant protein 1(MCP-1) were measured by Real-time fluorescent quantitative PCR and the concentrations of secreted TNF-α, IL-6, MIP-1α and MCP-1 were detected by ELISA. In addition, the expression of costimulatory molecules CD80, CD86, CD40 and histocompatibility complex II (MHC II) on the cell surface was analyzed by flow cytometry. Results After inducing by different TLR ligands, mRNA expression levels of inflammatory cytokines and chemokines were increased in both macrophages. The secretion of TNF-α, IL-6, MIP-1α and MCP-1 in peritoneal macrophages and the expression of CD86 and MHC II on the surface of peritoneal macrophages were significantly higher than those of bone marrow derived macrophages. Conclusion There are significant differences in the expression of inflammatory factors, chemokines, costimulatory molecules, and histocompatibility complex between bone marrow derived macrophages and peritoneal macrophages. Peritoneal macrophages have more complete macrophage function and is more suitable for immunological research and immunomodulatory drug evaluation.

The interaction between the costimulatory molecules CD80/86 and CD28 contributed to CD4+ T lymphocyte activation in flounder (Paralichthys olivaceus).

CD28 and CD80/86 are crucial co-stimulatory molecules for the T cell activation. Previous study illustrated that CD28 and CD80/86 present on T cells and antigen-presenting cells in flounder (Paralichthys olivaceus), respectively. The co-stimulatory molecules were closely associated with cell immunity. In this paper, recombinant protein of flounder CD80/86 (rCD80/86) and phytohemagglutinin (PHA) were added to peripheral blood leukocytes (PBLs) in vitro. Lymphocytes were significantly proliferated with CFSE staining, and the proportion of CD4+ and CD28+ lymphocytes significantly increased. In the meantime, genes related to the CD28-CD80/86 signaling pathway or T cell markers were significantly upregulated (p < 0.05). For further study, the interaction between CD80/86 and CD28 was confirmed. The plasmid of CD28 (pCD28-FLAG and pVN-CD28) or CD80/86 (pVC-CD80/86) was successfully constructed. In addition, pVN-ΔCD28 without the conserved motif "TFPPPF" was constructed. The results showed that bands of pCD28-FLAG bound to rCD80/86 were detected by both anti-FLAG and anti-CD80/86. pVN-CD28 complemented to pVC-CD80/86 showing positive fluorescent signals, and pVN-ΔCD28 failed to combine with pVC-CD80/86. The motif "TFPPPF" in CD28 played a crucial role in this linkage. These results indicate that CD28 and CD80/86 molecules interact with each other, and their binding may modulate T lymphocytes immune response in flounder. This study proved the existence of CD28-CD80/86 signaling pathway in flounder.

Co-stimulatory pathway competitive assay development using Liquid chromatography-tandem mass spectrometry (LC-MS/MS).

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Impaired monocyte-derived dendritic cell phenotype in prostate cancer patients: A phenotypic comparison with healthy donors.

BACKGROUND: Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte-derived DCs (Mo-DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo-DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD-DCs). METHODS: Mo-DCs were isolated from PCA patient samples, and their phenotype was compared to HD-DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA-DR, CD80, CD86) were assessed. RESULTS: PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA-DCs expressed significantly lower levels of maturation markers, including HLA-DR, CD80, and CD86, when compared to HD-DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). CONCLUSION: The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo-DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo-DCs for more effective cancer immunotherapies.

Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment.

BACKGROUND: Alpha-2-glycoprotein 1, zinc-binding (ZAG), a secreted protein encoded by the AZGP1 gene, is structurally similar to HLA class I. Despite its presumed immunological function, little is known about its role in tumor immunity. In this study, we thus aimed to determine the relationship between the expression of AZGP1/ZAG and the immunological profiles of breast cancer tissues at both the gene and protein level. METHODS: Using a publicly available gene expression dataset from a large-scale breast cancer cohort, we conducted gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1. We analyzed the correlation between AZGP1 expression and immune cell composition in breast cancer tissues, estimated using CIBERSORTx. Previously, we evaluated the infiltration of 11 types of immune cells for 45 breast cancer tissues using flow cytometry (FCM). ZAG expression was evaluated by immunohistochemistry on these specimens and analyzed for its relationship with immune cell infiltration. The action of ZAG in M1/M2 polarization models using primary cultures of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) was analyzed based on the expression of M1/M2 markers (CD86, CD80/CD163, MRC1) and HLA class I/II by FCM. RESULTS: AZGP1 expression was negatively correlated with multiple immunological processes and specific immune cell infiltration including Mφ M1 using GSEA and CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in tumor tissues assessed using FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I/II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. CONCLUSION: ZAG is suggested to be a novel immunoregulatory factor affecting the Mφ phenotype in breast cancer tissues.

PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice.

Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhanced spontaneous priming of thymus-derived (FOXP3+Helios+) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded tumor control by a simultaneous, RT-induced CD8+ cytotoxic T cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 (PD-L1+CD80+), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the control of such tumors by enabling a CTL response.

Absence of CD80 reduces HSV-1 replication in the eye and delays reactivation but not latency levels.

Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80-/- mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80-/- mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

Costimulatory receptors in the channel catfish: CD28 family members and their ligands.

The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (Ictalurus punctatus), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of cd28, ctla4, cd80/86, b7h1/dc, b7h3, b7h4, b7h5, two btla, and four b7h7 genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.

The role of TRIF protein in regulating the proliferation and antigen presentation ability of myeloid dendritic cells through the ERK1/2 signaling pathway in chronic low-grade inflammation of intestinal mucosa mediated by flagellin-TLR5 complex signal.

Objective: The objective is to explore whether the flagellin-TLR5 complex signal can enhance the antigen presentation ability of myeloid DCs through the TRIF-ERK1/2 pathway, and the correlation between this pathway and intestinal mucosal inflammation response. Methods: Mouse bone marrow-derived DC line DC2.4 was divided into four groups: control group (BC) was DC2.4 cells cultured normally; flagellin single signal stimulation group (DC2.4+CBLB502) was DC2.4 cells stimulated with flagellin derivative CBLB502 during culture; TLR5-flagellin complex signal stimulation group (ov-TLR5-DC2.4+CBLB502) was flagellin derivative CBLB502 stimulated ov-TLR5-DC2.4 cells with TLR5 gene overexpression; TRIF signal interference group (ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA) was ov-TLR5-DC2.4 cells with TLR5 gene overexpression stimulated with flagellin derivative CBLB502 and intervened with TRIF-specific inhibitor Pepinh-TRIFTFA. WB was used to detect the expression of TRIF and p-ERK1/2 proteins in each group of cells; CCK8 was used to detect cell proliferation in each group; flow cytometry was used to detect the expression of surface molecules MHCI, MHCII, CD80, 86 in each group of cells; ELISA was used to detect the levels of IL-12 and IL-4 cytokines in each group. Results: Compared with the BC group, DC2.4+CBLB502 group, and ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, the expression of TRIF protein and p-ERK1/2 protein in ov-TLR5-DC2.4+CBLB502 group was significantly upregulated (TRIF: p = 0.02,  = 0.007,  = 0.048) (ERK1: p < 0.001, =0.0003,  = 0.0004; ERK2:p = 0.0003,  = 0.0012,  = 0.0022). The cell proliferation activity in ov-TLR5-DC2.4+CBLB502 group was enhanced compared with the other groups (p = 0.0001, p < 0.0001, p = 0.0015); at the same time, the expression of surface molecules MHCI, MHCII, CD80, 86 on DCs was upregulated (p < 0.05); and the secretion of IL-12 and IL-4 cytokines was increased, with significant differences (IL-12: p < 0.0001, p < 0.0001, p = 0.0005; IL-4: p =  < 0.0001, p =  < 0.0001, p = 0.0001). However, the ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, which was treated with TRIF signal interference, showed a decrease in intracellular TRIF protein and p-ERK1/2 protein, as well as a decrease in cell proliferation ability and surface stimulation molecules, and a decrease in the secretion of IL-12 and IL-4 cytokines (p < 0.05). Conclusion: After stimulation of flagellin protein-TLR5 complex signal, TRIF protein and p-ERK1/2 protein expression in myeloid dendritic cells were significantly up-regulated, accompanied by increased proliferation activity and maturity of DCs, enhanced antigen presentation function, increased secretion of pro-inflammatory cytokines IL-12 and IL-4. This process can be inhibited by the specific inhibitor of TRIF signal, suggesting that the TLR5-TRIF-ERK1/2 pathway may play an important role in abnormal immune response and mucosal chronic inflammation infiltration mediated by flagellin protein in DCs, which can provide a basis for our subsequent animal experiments.

A better understanding of the role of the CTLA-CD80/86 axis in the treatment of autoimmune diseases.

Autoimmune diseases are diseases in which the regulatory mechanisms of the immune response are disturbed. As a result, the body loses self-tolerance. Since one of the main regulatory mechanisms of the immune response is the CTLA4-CD80/86 axis, this hypothesis suggests that autoimmune diseases potentially share a similar molecular basis of pathogenesis. Hence, investigating the CTLA4-CD80/86 axis may be helpful in finding an appropriate treatment strategy. Therefore, this study aims to investigate the molecular basis of the CTLA4-CD80/86 axis in the regulation of the immune response, and then its role in developing some autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. As well, the main therapeutic strategies affecting the CTLA4-CD80/86 axis have been summarized to highlight the importance of this axis in management of autoimmune diseases.

Clinicopathologic Evaluation of CD80, CD86, and PD-L1 Expressions with Immunohistochemical Methods in Malignant Melanoma Patients.

OBJECTIVE: Diagnostic and prognostic biomarkers for malignant melanoma are crucial for treatment and for developing targeted therapies. Malignant melanoma is a highly immunogenic tumor, and its regression, treatment, and prognostic evaluation are directly related to escape from immune destruction. Therefore, we aimed to determine the expression levels of CD80, CD86, and PD -L1 in malignant melanoma tissue samples by immunohistochemistry and to investigate the possible relationship between these proteins and the clinicopathological features in this study. MATERIAL AND METHODS: Hematoxylin and eosin staining and immunohistochemical staining for CD80, CD86, and PD-L1 were evaluated for clinical data, survival, prognosis, tumor location, malignant melanoma subtypes, tumor size, and prognostic findings. RESULTS: Higher survival rates were observed in patients with lower PD-L1 staining scores in the tumor. The 5-year survival was higher in patients with CD80-positive and CD86-positive biopsies. Mortality was lower in superficial spreading melanoma and Lentigo maligna melanoma types, whereas staining positivity of CD80 and CD86 was higher. Furthermore, a relationship between clinical stage and Breslow thickness ( < 2mm/≥2mm), tumor ulceration, lymph node metastasis, and CD80 and CD86 expression was also identified. CONCLUSION: Our findings suggest that PD-L1, CD80, and CD86 expression are essential in malignant melanoma and could be used as prognostic markers.

Causal role of immune cells in chronic obstructive pulmonary disease: Mendelian randomization study.

OBJECTIVES: Innate and adaptive immunity play different roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, previous studies on the relationship between immune cells and COPD reported inconsistent results. METHODS: The causal connection between 731 immune cells and COPD was established using a two-sample Mendelian randomization (MR) analysis through publicly accessible genetic data. The heterogeneity and horizontal pleiotropism of the findings were confirmed using sensitivity analysis. RESULTS: In the B-cell panel, B-cell activating factor receptor (BAFF-R) on CD20- and CD20 on IgD-CD38bright (OR (95% CI): 0.93 (0.88, 0.99) and 0.97 (0.95, 0.98), respectively) were discovered to be protective. In the cDC panel, CD62L- plasmacytoid DC AC, CD80 on monocytes and CD11c on myeloid DCs (OR (95% CI): 0.94 (0.92, 0.97), 0.97 (0.94, 0.99) and (0.97 (0.95, 0.98), respectively) exerted protective effects. However, unswitched memory AC (OR (95%CI): 1.08 (1.01,1.15)) and CD 19 on IgD- CD 27- (OR (95%CI): 1.06 (1.02,1.10)) were hazardous in the B-cell panel. However, among the 731 immune cell phenotypes, no causal relationship was found for COPD on immune cells. CONCLUSION: This study found a potential causal relationship between immune cells in COPD, ruling out reverse causation. This study provides new avenues for studying the mechanisms of COPD.

Patients with primary focal segmental glomerulosclerosis with detectable urinary CD80 are more similar to patients with minimal change disease in clinicopathological features.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is an important cause of refractory nephrotic syndrome (NS) in children and adults. Urinary CD80 is elevated in some patients with primary FSGS, however, its clinical value is not fully clarified. This study aims to evaluate the clinical and pathological significance of urinary CD80 in patients with primary FSGS. METHODS: Sixty-one adult patients with biopsy-proven primary FSGS, with standard treatment and long-term follow up, were enrolled retrospectively. Urinary CD80, on the day of kidney biopsy, was measured using commercial ELISA kits and adjusted by urinary creatinine excretion. Their associations with clinical and pathological parameters were investigated. RESULTS: Urinary CD80 was detectable in 30/61 (49.2%) patients, who presented with a higher level of proteinuria (10.7 vs. 5.8 g/24h; p = 0.01), a lower level of serum albumin (19.3 ± 3.9 vs. 24.2 ± 8.2 g/L; p = 0.005), a higher prevalence of hematuria (70.0 vs. 38.7%; p = 0.01), and showed a lower percentage of segmental glomerulosclerosis lesion [4.8 (3.7-14.0) vs. 9.1 (5.6-21.1) %; p = 0.06]. The cumulative relapse rate was remarkably high in these patients (log-rank, p = 0.001). Multivariate analysis identified that the elevated urinary CD80 was an independent risk factor for steroid-dependent NS (OR 8.81, 95% CI 1.41-54.89; p = 0.02) and relapse (HR, 2.87; 95% CI 1.29-6.38; p = 0.01). CONCLUSIONS: The elevated urinary CD80 is associated with mild pathological change and steroid-dependent cases of primary FSGS adults, which indicates these patients are more similar to minimal change disease (MCD) in clinicopathological features.

Immunomodulatory effects of nanoparticles on dendritic cells in a model of allergic contact dermatitis: importance of PD-L2 expression.

Nanoparticle (NP) skin exposure is linked to an increased prevalence of allergic contact dermatitis. In our prior studies using the mouse contact hypersensitivity (CHS) model, we reported that silica 20 nm (SiO2) NPs suppressed the allergic response and titanium dioxide NPs doped with manganese (mTiO2) exacerbated it. In this work, we conducted in vitro experiments using bone marrow-derived dendritic cells (BMDCs) to study the combinatorial effect of the potent 2,4-dinitrofluorobenzene (DNFB) hapten sensitizer with SiO2 and mTiO2 NPs on BMDC cytotoxicity, cytokine secretion and phenotype using the B7 family ligands. Results show that DNFB and mTiO2 behave similarly and exhibit proinflammatory characteristics while SiO2 promotes a naive phenotype. We observe that the B7-H3 (CD276) ligand is only expressed on CD80 + (B7-1) BMDCs. Results from adoptive transfer CHS studies, combined with BMDC phenotype analysis, point to the importance of PD-L2 expression in modulating the adaptive immune response. This work identifies metrics that can be used to predict the effects of NPs on contact allergy and to guide efforts to engineer cell-based therapies to induce hapten specific immune tolerance.

The Immunological Capacity of Thrombocytes.

Thrombocytes are numerous in the blood of aves (birds) and ichthyoids (fish). The origin of this cell type is a common hematopoietic stem cell giving rise to a cell that is active in blood coagulation, inflammatory functions, and the immune response in general. It has been well documented that thrombocytes can phagocytize small particles and bacteria. While phagocytosis with an associated oxidative burst has been reported for chicken thrombocytes, some questions remain as to the degradation capacity of phagosomes in ichthyoids. As innate cells, thrombocytes can be stimulated by bacterial, viral, and fungal pathogens to express altered gene expression. Furthermore, there have been observations that led researchers to state that platelets/thrombocytes are capable of serving as "professional antigen presenting cells" expressing CD40, CD80/86, MHC I, and MHC II. This indeed may be the case or, more likely at this time, provide supporting evidence that these cells aid and assist in the role of professional antigen-presenting cells to initiate adaptive immune responses.

Interleukin 6 exacerbates the progression of warm autoimmune hemolytic anemia by influencing the activity and function of B cells.

To explore the effect of IL-6 on the activity and secretory function of B cells and analyze its effect on clinical indicators and efficacy in wAIHA patients. This study included 25 hemolytic wAIHA patients, 13 remission patients, and 10 HCs. Plasma levels of various cytokines were detected using CBA. PBMCs were extracted from 12 hemolytic wAIHA patients and divided into three wells, stimulation with IL-6 and IL-6 + tocilizumab, the blank control wells were also set. After 48 h of in vitro cell culture, percentage of CD5+CD80+, CD5-CD80+,CD5+CD86+,CD5-CD86+,CD5+IL-10+,CD5-IL-10+B cells were determined by flow-cytometry. Plasma levels of IL-6 and IL-10 in hemolytic episode group were significantly higher than that in HCs group (p = 0.0243; p = 0.0214). RBC and Hb levels were negatively correlated with IL-6 levels in wAIHA patients, while LDH levels were positively correlated.Therapeutic effects of glucocorticoid and duration of efficacy were also significantly correlated with IL-6 levels in wAIHA patients. After 48 h in vitro cell culture, percentages of CD80+/CD5+CD19+and CD80+/CD5-CD19+ cells in the IL-6 stimulation group were higher than those in blank control group (p = 0.0019; p = 0.0004), while CD86+/CD5+ CD19+ and CD86+/CD5-CD19+ cells were not statistically different before and after IL-6 stimulation. Percentage of IL-10+/CD5+ CD19+ cells in IL-6 stimulation group was lower than that in blank control (p = 0.0017) and IL-6 + toc (p = 0.0117) group. Percentage of IL-10+/CD5- CD19+cells in the IL-6 stimulation group was lower than that in the blank control group (p = 0.0223). Plasma levels of IL-6 were significantly elevated in hemolytic wAIHA patients and correlated with clinical indicators and efficacy. IL-6 promotes the activation of B cells. Although the results were not statistically significant, IL-6R antagonist tocilizumab may hopefully become a targeted therapy for wAIHA patients.

Quantitative analysis of soluble costimulatory molecules as potential diagnostic biomarkers for rheumatoid arthritis using LC-MS/MS in MRM mode.

BACKGROUND AND AIMS: Rheumatoid arthritis (RA) is a chronic autoimmune disease. RA-induced immunological responses are coordinated by T-cell stimulation. The costimulatory signal CD28-B7 is essential for T-cell activation by interacting CD28 with CD80 and CD86 costimulatory proteins. CTLA4 is another costimulatory protein that binds to CD80 and CD86 to inhibit T-cell activity. The soluble costimulatory proteins: sCD80, sCD86, sCD28, and sCTLA-4 were detected and quantified in human plasma and correlated with RA development. As potential diagnostic biomarkers for RA, developing a sensitive, specific, and reproducible method for quantifying these costimulatory molecules in human plasma and establishing quantitative ranges for each protein in healthy and RA patients' plasma is essential for advancing the clinical diagnostic and health outcomes. MATERIALS AND METHODS: A novel quantitative liquid chromatography-tandem spectrometry (LC-MS/MS) technique using multiple reaction monitoring (MRM) modes was developed and validated to measure soluble costimulatory molecules sCTLA4, sCD28, sCD80, and sCD86 in human plasma samples. Furthermore, the method was applied to determine sCTLA4, sCD28, sCD80, and sCD86 levels in plasma samples from RA patients (n = 23) and healthy controls (n = 21). RESULTS: The method was successfully developed and validated according to international inter- and intra-assay precision and accuracy guidelines. The linearity of the method was achieved between 0.5 nM and 100 nM for each protein with a correlation coefficient of > 0.998. The plasma level of sCTLA4, sCD80, and sCD86 in RA patients was significantly elevated compared to controls. RA patients had 63.32 ± 17.63 nM sCTLA4 and controls 36.05 ± 18.83 nM; p < 0.0001. The performance of the four proteins was determined using ROC curves, where sCTLA4 showed the highest diagnostic and clinical performance compared to the others. CONCLUSIONS: This study reports the first use of LC-MS/MS in MRM mode to accurately quantify soluble costimulatory molecules in plasma samples as potential RA diagnostic biomarkers. Determination of the reference range for each protein with high selectivity and sensitivity increases the potential for utilizing this method as a clinical diagnostic.

Photodynamic therapy changes tumour immunogenicity and promotes immune-checkpoint blockade response, particularly when combined with micromechanical priming.

Photodynamic therapy (PDT) with redaporfin stimulates colon carcinoma (CT26), breast (4T1) and melanoma (B16F10) cells to display high levels of CD80 molecules on their surfaces. CD80 overexpression amplifies immunogenicity because it increases same cell (cis) CD80:PD-L1 interactions, which (i) disrupt binding of T-cells PD-1 inhibitory receptors with their ligands (PD-L1) in tumour cells, and (ii) inhibit CTLA-4 inhibitory receptors binding to CD80 in tumour cells. In some cancer cells, redaporfin-PDT also increases CTLA-4 and PD-L1 expressions and virtuous combinations between PDT and immune-checkpoint blockers (ICB) depend on CD80/PD-L1 or CD80/CTLA-4 tumour overexpression ratios post-PDT. This was confirmed using anti-CTLA-4 + PDT combinations to increase survival of mice bearing CT26 tumours, and to regress lung metastases observed with bioluminescence in mice with orthotopic 4T1 tumours. However, the primary 4T1 responded poorly to treatments. Photoacoustic imaging revealed low infiltration of redaporfin in the tumour. Priming the primary tumour with high-intensity (~ 60 bar) photoacoustic waves generated with nanosecond-pulsed lasers and light-to-pressure transducers improved the response of 4T1 tumours to PDT. Penetration-resistant tumours require a combination of approaches to respond to treatments: tumour priming to facilitate drug infiltration, PDT for a strong local effect and a change in immunogenicity, and immunotherapy for a systemic effect.

Immune checkpoint activity regulates polycystic kidney disease progression.

Innate and adaptive immune cells modulate the severity of autosomal dominant polycystic kidney disease (ADPKD), a common kidney disease with inadequate treatment options. ADPKD has parallels with cancer, in which immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have previously shown that in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte associated protein 4 (CTLA-4) on T cells and programmed cell death ligand 1 (PD-L1)/CD80 on macrophages and epithelial cells in Pkd1RC/RC mice versus WT, paralleling disease severity. PD-L1/CD80 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking 2 immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD severity. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers correlative with PKD severity. Together, our data suggest that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.